Since cancer is fundamentally a human problem, it is critical to understand the process as related to the human species. Studies on the mechanisms of rodent cell malignant transformation in vitro have been elegantly carried out and allow detailed analysis of the process of carcinogenesis in these species. Unfortunately, the malignant transformation of human cells in culture does not occur spontaneously or as a result of carcinogen treatment, despite intensive work by many investigators, including our group. The few reports of the malignant transformation of human cells in vitro that have been published seem to involve a large element of chance. However, it is now possible to induce human fibroblasts to express all but one of the common phenotypic changes characteristic of the transformed rodent fibroblasts. The property of unlimited lifespan (UL) often referred to as "immortality" is never seen. This may be because rodent fibroblasts and human fibroblasts are fundamentally different, perhaps reflecting the great difference in lifespan of these species. As discussed in the proposal, there are reasons to believe that this is not true. It seems much more likely that the failure to obtain a UL human fibroblast is an artifact of cell culture. Therefore, this proposal is an all out effort to develop methods to reproducibly induce UL human fibroblasts. At the same time, three diploid UL human fibroblast cell lines, the only ones know to us, will be used to determine the role of various other changes in the multistepped process of malignant transformation. Using fibroblast cell lines derived from normal persons and from patients with Bloom's syndrome and Fanconi's anemia, we will try to induce cells with unlimited lifespans. To do so, we will grow the cells under conditions of increased oxygen tension to induce clastogenic effects. We will also expose normal fibroblasts repeatedly to the anti 7,8-diol-9,10-epoxide of benzo(a)pyrene because exposure of Syrian hamster fibroblasts to this carcinogen led to UL. We will select these series of treated populations for nonsenescing cells, using conditions that avoid metabolic cooperation between senescing and nonsenescing cells. While the above experiments are in progress we will expose three fibroblast cell lines that have already acquired unlimited lifespan (HUT 11, HUT, 12 and LGIvmyc1) to carcinogens in order to induce them to acquire the rest of the series of tumorcellrelated characteristics in a stepwise fashion and determine which combination of traits confers on them the ability to form tumors. We will also determine how fibroblasts derived from human fibromas and low grade maligancies differ from normal cells and from fibrosarcomaderived cells since they may represent intermediates on the path to malignancy.